A method to prepare macromolecular particles embedded in vitreous ice was initially described in Adrian et al., 1984 and Dubochet et al., 1985, and since then thin samples have continued to be made by blotting excess liquid from electron microscopy grids with filter paper. In many cases, it is likely that evaporation plays a significant role in thinning the sample, and often this has a detrimental effect on the structure of the specimen. The use of computerized control of key parameters (such as, for example, the ambient temperature and humidity, the blotting pressure and duration, and the interval between blotting and final vitrification) has improved the sophistication and the reproducibility with which cryo-specimens can be prepared. Even so, the results still remain less consistent from trial to trial and over the entire area of an electron microscopy grid. In particular, preparation of specimens at a desired thickness of ˜30 nanometers (nm) to 50 nm remains unreliable.
Achieving specimen thicknesses well below ˜100 nm becomes increasingly important as the resolution of cryo-EM images is increased (Agard et al., 2014). This is because a single image may contain particles located at different focal heights if the vitrified ice is significantly thicker than the particle size. Merging such data leads to an unwanted envelope function (Jensen, 2001) that is equivalent to the one produced by varying the focus of the objective lens by the same amount.
Even if all particles are tethered at a common Z-height, as is possible when using a continuous support film, an ice-film thickness significantly greater than the size of the particle will necessarily cause an increase in the fraction of electrons that are inelastically scattered. This is undesirable because any unnecessary increase in the fraction of inelastically scattered electrons leads to a corresponding loss of useful signal in the image.